A variety of autoimmune diseases have been associated with serologically defined variants of the human leukocyte antigen (HLA) class III antigens. The HLA region, located on the short arm of chromosome 6, encodes many different glycoproteins that have been classified into two categories. The first category, class I products, encoded by the HLA-A, -B, and -C loci, are on the surface of all nucleated cells and function as targets in cytolytic T-cell recognition. The second category, class II products, encoded by the HLA-D region, are involved in cooperation and interaction between cells of the immune system. These class II products appear to be encoded by at least three distinct loci, DR, DQ and DP, each with its distinct alpha and beta chains. The class II loci of the human major histocompatibility complex (MHC) encode highly polymorphic cell-surface glycoproteins (macrophage and .beta.-cell transmembrane glycoproteins). [For review article, see Giles et al. Adv. in Immunol. 37:1-71 (1985).] The polymorphism in class II antigens is localized to the NH.sub.2 -terminal outer domain and is encoded by the second exon. The class II polymorphic residues have been postulated to interact with the T-cell receptor or with foreign antigen or both [Sette et al., Nature, 328:395-399 (1987)] with recognition of the antigen peptide fragments in association with a specific class II product leading to T cell activation and consequent stimulation of antibody production by .beta. lymphocytes [Marx et al., Science, 238:613-614 (1987).]
This invention relates to HLA class II beta genes and proteins associated with autoimmune diseases and methods for their diagnostic detection. Specifically, the autoimmune diseases on which this invention focuses are insulin-dependent diabetes mellitus (IDDM) and Pemphigus vulgaris (PV).
Insulin-dependent diabetes mellitus (IDDM), a chronic autoimmune disease also known as Type I diabetes, is a familial disorder of glucose metabolism susceptibility associated with specific allelic variants of the human leukocyte antigens (HLA). The dysfunctional regulation of glucose metabolism occurring in IDDM patients results from the immunologically mediated destruction of the insulin-producing islet cells of the pancreas, the beta cells. The development of IDDM can be divided into six stages, beginning with genetic susceptibility and ending with complete destruction of beta-cells. G. Eisenbarth, N. Eng. J. Med., 314:1360-1368 (1986). [Donaich et al., Annu. Rev. Med., 34:13-20 (1983).]. More than 90% of all IDDM patients carry the DR3 and/or DR4 antigen, and individuals with both DR3 and DR4 are at greater risk than individuals who have homozygous DR3/3 or DR4/4 genotypes. L. Raffel and J. Rotter, Clinical Diabetes, 3:50-54 (1985); Svejgaard et al., Immunol. Rev., 70:193-218 (1983); L. Ryder et al., Ann. Rev. Genet., 15:169-187 (1981).
Pemphigus derived from the Greek pemphix meaning blister or pustule is the name applied to a distinctive group of chronic or acute skin diseases characterized by successive crops of itching bullae. Pemphigus vulgaris (PV) is a rare relapsing disease manifested by suprabasal, intraepidermal bullae of the skin and mucus membranes, which is invariably fatal if untreated; however, remission has been obtained by the use of corticosteroid hormones and immunosuppressive drugs. PV, an autoimmune disease, has been strongly associated with the HLA serotypes DR4 and DRw6 [Brautbar et al., Tissue Antigens, 16:238-241 (1986)] with less than 5% of PV patients possessing neither marker. Disease associations with two different haplotypes can be interpreted to mean (1) the two haplotypes share a common allele or epitope, or, alternatively, that (2) different alleles on the two haplotypes are capable of conferring disease susceptibility.
Molecular analysis of the HLA class II genes has revealed that the HLA serotypes are genetically heterogeneous, and that, in particular, the DR4 haplotype consists of five different DR.beta.I allelic sequences corresponding to the five mixed lymphocyte culture (MLC) defined types Dw4, Dw10, Dw13, Dw14 and Dw15 [Gregersen et al., PNAS (USA) 83:2642-2646 (1986)] and three different DQ.beta. allelic sequences corresponding to the DQ.beta.3.1, DQ.beta.3.2 and DQ-blank types [Erlich et al., in Schacter et al. (eds.): The Molecular Analysis of Histocompatibility Antigens, pp. 93-109 (1987)]. Virtually all of the extensive polymorphism characteristic of the class II loci has been localized to the second exon.
Sequence analysis of coding sequence polymorphisms in the DR.beta. loci revealed that the sequence or epitope in the DR4 DR.beta.I chain that distinguishes Dw10 from the other DR4 subtypes is shared by the DR.beta.I chain of the DRw6 haplotype. [Gorski et al., Nature, 322:67-70 (1986)]. Recently, restriction fragment length polymorphisms (RFLPs) which subdivide the DR4 and DRw6 haplotypes were obtained by using a HLA-DQ.beta. cDNA probe; such RFLPs have been reported to be even more highly associated with PV than are the serologic markers [Szafer et al., 1987, Proc. Natl. Acad. Sci. USA 84:6542-6545.].
Of all the immunologically defined polymorphisms, the HLA-DR beta region has been found to be most strongly associated with IDDM. Therefore, restriction fragments of the HLA class II-.beta. DNA have been analyzed for use as genetic markers of insulin-dependent diabetes mellitus. D. Owerbach et al., Diabetes, 33:958-964 (1984); O. Cohen-Haguenauer et al., PNAS (USA), 82:3335-3339 (1985); D. Stetler et al., PNAS (USA), 82:8100-8104 (1985).
Arnheim et al., PNAS (USA), 82:6970-6974 (October 1985), examined DNA polymorphisms within the HLA class II loci associated with susceptibility to IDDM by using genomic blot-hybridization analysis with DQ.beta. and DR.beta. cDNA probes. Described therein is a DQ.beta. subdivision of the DR4 haplotype wherein one DR4 variant had a RsaI restriction fragment length polymorphism (RFLP) of 1.8 kb and another had a RsaI RFLP of 1.5 kb. The DQ.beta.-related 1.5 kb RsaI fragment was reported to identify a number of non-DR4 IDDM individuals as well as 90% of all IDDM DR4 individuals.
Other investigators using other restriction enzymes (e.g., BamHI, HindIII) have reported RFLP subdivisions of the DR4 haplotype using DQ.beta. probes. [Holbeck et al., Immunogenetics (1986) 24:251-258; Henson et al., Immunogenetics, (1987) 25:152-160).] Holbeck et al., id., found that the RFLP subsets of DR4, designated DQw3.1 and DQw3.2 are distinguishable by the reactivity of their expressed products with a specific monoclonal antibody TA10. [Kim et al., PNAS (USA), 82:8139-8142 (1985); Tait et al., Tissue Antigens, (1986) 28:65-71.] The DQw3.1 subtype correlates with the serologic specificity TA10.sup.+, whereas DQw3.2 correlates with TA10.sup.-.
Now abandoned U.S. Ser. No. 899,344 (filed Aug. 22, 1986) entitled "Process for Detecting Specific Nucleotide Variations and Genetic Polymorphisms Present in Nucleic Acids" (incorporated herein by reference) discloses the cloning and sequencing of the RsaI 1.5 kb (DQw3.2) and the RsaI 1.8 kb (DQw3.1) variants of DR4 haplotypes and illustrates the differences in the sequences thereof. (Such differences are shown herein in Tables III and IV.)
WO 86/07464 discloses a specific DQ.beta..sub.2 allelic variant, DQw3.2 as a specific genomic marker associated with IDDM, and provides two methods of identifying individuals at increased risk of diabetes. The first method involves the use of a labeled probe to detect the DQw3.2 allele, whereas the second method involves the serologic detection of the DQw3.2 allele.
Erlich et al., in Schacter et al., (eds.), Perspectives in Immunogenetics and Histocompatibility, Vol. 7:93-106 (1987), reported the protein translation sequences for the DQw3.1 and 3.2 variants.
Michelson et al., J. Clin. Invest., 79:1144-1152 (April 1987), reported the nucleotide sequence for the DQw3.1 variant.
Acha-Orbea et al., PNAS (USA), 84(8):2435-2435 (1987) reported on differences in the H-2 I-A region of control mice and diabetes-susceptible NOD (non-obese diabetic) mice. Normal mice have an aspartate residue at position 57 of said region whereas NOD mice have a neutral serine residue at that position. The human HLA-DQ.beta. region is analogous to the H-2 I-A region of the mouse.
Yoon et al., Diabetes Care, 8 (suppl. 1):39-44 (September-October 1985), presents a review of the evidence for viruses as a trigger for IDDM in animals and humans. See also, Bodansky et al., Lancet, (1986), ii:1351-1353; Kagnoff et al., J. Exp. Med., (1984), 160:1544-1557; McChesney et al., Ann. Rev. Immunol. (1987), 5:279-304; Oldstone et al., in Notkins et al. (eds.), Concepts in Viral Pathogenesis (1986); Schwimmback et al., J. Exp. Med. (1987), 166:173-181; Silver et al., Disease Markers (1985), 3:155-168; and Srinivasappa et al., J. Virol., 57:397-401.
Roudier et al., Abstract from the American Rheumatism Association (Western Region) meeting in San Diego, Calif., Nov. 5-7, 1987, reported that the HLA Dw4 DR.beta.1 chain and an Epstein-Barr virus (EBV) glycoprotein share a hexapeptide.
Todd et al., Nature, 329:599-604 (Oct. 15, 1987) discusses the contribution of the HLA DQ.beta. gene to susceptibility and resistance to IDDM. The authors conclude that "the structure of the DQ molecle, in particular residue 57 of the .beta.-chain, specifies the autoimmune response against the insulin-producing islet cells."
Many HLA DR.beta. sequences have been published previously. The sequence AspIleLeuGluAspGluArg was reported by Gregersen et al., PNAS (USA), 83:2642-2646 (1986) as part of a study of the diversity of DR.beta. genes from HLA DR4 haplotypes. No mention was made of an association thereof with diabetes. In addition, J. Gorski and B. Mach, Nature, 322:67-70 (1986) reported on HLA-DR polymorphism within a group including the haplotypes DR3, DR5 and DRw6. The nucleotide sequences found in the polymorphic regions at the .beta.I locus were not discussed regarding association with diabetes. The first publication on HLA sequences from diabetics is that by D. Owerbach et al., Immunogenetics, 24:41-46 (1986). This paper is based on the study on a HLA-DR.beta. gene library from one IDDM patient. The analysis of class II polymorphism and disease susceptibility requires the comparison of many sequences derived from patients and HLA-matched controls.
Allelic variation in the class II antigens is restricted to the outer domain encoded by the second exon of the protein. Serologic methods for detecting HLA class II gene polymorphism are not capable of detecting much of the variation detectable by DNA methods.
Allelic variations may be detected independently of restriction site polymorphism by using sequence-specific synthetic oligonucleotide probes. Conner et al., PNAS (USA), 80:278 (1983). This technique has been applied to study the polymorphism of HLA DR-.beta. using Southern blotting. Angelini et al., PNAS (USA), 83:4489-4493 (1986).
A further refinement of the technique using sequence-specific oligonucleotide probes involves amplifying the nucleic acid sample being analyzed using selected primers, four nucleotide triphosphates, and an appropriate enzyme such as DNA polymerase, followed by detecting the nucleotide variation in sequence using the probes in a dot blot format, as described in now abandoned Ser. No. 899,344, supra, and in now abandoned Ser. No. 839,331 filed Mar. 13, 1986. A temperature cycling process wherein a thermostable enzyme is added only once in the amplification process is described in now abandoned Ser. No. 899,513 and Ser. No. 063,647 now U.S. Pat. No. 4,965,188, filed respectively Aug. 22, 1986 and Jun. 17, 1987, both entitled "Process for Amplifying, Detecting, and/or Cloning Nucleic Acid Sequences Using a Thermostable Enzyme." Now abandoned Ser. No. 899,241 and U.S. Pat. No. 4,889,818, filed respectively Aug. 22, 1986 and Jun. 17, 1987, both entitled "Purified Thermostable Enzyme" disclose and claim thermostable enzymes, purified or recombinant, which can be used in said amplification process.
There is a need in the art for subdivision of the serologic markers HLA DR3, DR4 and DRw6 to obtain more informative and more precisely defined markers for susceptibility to the autoimmune diseases IDDM and PV. Further, there is a need in the art to identify susceptibility conferring haplotypes which are neither DR3, DR4 nor DRw6.
Previously, the distinction between the IDDM associated DQ.beta. variants, DQw3.1 and DQw3.2, of the DR4 haplotype has been made by RFLP or by the use of antibodies. This invention in one aspect relates to methods to identify such DQ.beta. variants.